1. Field of the Invention
This invention relates to compositions and methods for delivering bioactive agents to cells. In preferred embodiments, this invention relates to cationic lipopolymers comprising a polyethylenimine (PEI), a biodegradable group, and a relatively hydrophobic group, and to methods of using such lipopolymers to deliver bioactive agents such as DNA, RNA, oligonucleotides, proteins, peptides, and drugs.
2. Description of the Related Art
A number of techniques are available for delivering bioactive agents to cells, including the use of viral transfection systems and non-viral transfection systems. Viral systems typically have higher transfection efficiency than non-viral systems, but there have been questions regarding the safety of viral systems. See Verma I. M and Somia N., Nature 389 (1997), pp. 239-242; Marhsall E. Science 286 (2000), pp. 2244-2245. In addition, viral vector preparation tends to be a complicated and expensive process. Although non-viral transfection systems generally are less efficient than viral systems, they have received significant attention because they are generally believed to be safer and easier to prepare than viral systems.
A number of non-viral transfection systems involve the use of cationic polymers that are complexed to bioactive agents. Examples of cationic polymer that have been used as gene carriers include poly(L-lysine) (PLL), polyethyleneimine (PEI), chitosan, PAMAM dendrimers, and poly(2-dimethylamino)ethyl methacrylate (pDMAEMA). Unfortunately, transfection efficiency is typically poor with PLL, and high molecular weight PLL has shown significant toxicity to cells. In some cases PEI provides efficient gene transfer without the need for endosomolytic or targeting agents. See Boussif O., Lezoualc'h F., Zanta M. A., Mergny M. D., Scherman D., Demeneix B., Behr J. P., Proc Natl Acad Sci USA. Aug. 1, 1995, 92(16) 7297-301. A range of polyamidoamine dendrimers have been studied as gene-delivery systems. See Eichman J. D., Bielinska A. U., Kukowska-Latallo J. F., Baker J. R. Jr., Pharm. Sci. Technol. Today 2000 July; 3(7):232-245. Unfortunately, both PEI and dendrimers have been reported to be toxic to cells, thus limiting the potential for using PEI as a gene delivery tool in applications to human patients. In addition, the cost of polyamidoamine dendrimers having commercially practical gene transfection efficiencies is relatively high.
Gene carriers made with degradable cationic polymers have been reported to transfer genes into mammalian cells with decreased cytotoxicity. See Lim Y. B., Kim S. M., Lee Y., Lee W. K., Yang T. G., Lee M. J., Suh H., Park J. S., J. Am. Chem. Soc., 123 (10), 2460-2461, 2001. Unfortunately, these degradable systems also exhibited lower gene transfer efficiency compared to non-degradable polymers. To improve the transfection efficiency of low molecular weight PEI, Gosselin et al. reported that higher molecular weight PEI could be obtained by using disulfide-containing linkers. See Gosselin, Micheal A., Guo, Menjin, and Lee, Robert J. Bioconjugate Chem. 2001. 12:232-245. PEI polymers made using dithiobis(succinimidylpropionate) (DSP) and dimethyl-3,3′-dithiobispropionimidate-2HCl (DTBP) showed comparable gene transfection efficiency and lower cytotoxicity. However, the disulfide-containing linkers are expensive, which makes large-scale preparation of this system difficult and undesirable. The polymers with disulfide-containing linkers are only degraded under reducing conditions, which limits polymer applications in other conditions.
Lynn, et al. have described a method of synthesizing biodegradable cationic polymers using diacrylates as linker molecules between cationic compounds. See Lynn, David A.; Anderson, Daniel G.; Putnam, David; and Langer, Robert. J. Am. Chem. Soc. 2001, 123, 8155-8156. However, the resulting polymers do not complex well with many bioactive agents. Synthesis of these polymers requires days to complete and the amount of effective product, which can be used in gene delivery, is low. More than one hundred cationic polymers were produced according to the methods of Lynn et al., but only two of these polymers showed effective gene transfection efficiency. These factors make the preparation of high molecular weight polymers by this method difficult to achieve.
Thus, there remains a need for cationic polymers that may be used to safely and efficiently facilitate the delivery of bioactive agents to cells.